The ability of animals to move depends on motoneurons sending appropriate signals to muscles. This ability is compromised by spinal cord injury and in diseases such as amyotrophic lateral sclerosis (ALS). Our long term goal is to use individually identified motoneurons in embryonic zebrafish as a model to understand normal motoneuron development; this knowledge is critical for designing therapies for spinal cord injuries and diseases such as ALS. We hypothesize that transcription factor networks control specific aspects of motoneuron development such as axon morphology, neurotransmitter activity, expression of ion channels and receptors, and survival. We propose a series of experiments to address this hypothesis. Motoneurons do not form in the absence of the Isletl transcription factor, suggesting that it is a motoneuron "master regulator". In zebrafish lacking Isletl, motoneurons develop an interneuron-like morphology, but maintain expression of several motoneuron genes, including transcription factors. These results suggest that Isletl regulates some but not all aspects of motoneuron differentiation, and thus is not a motoneuron master regulator. We propose that motoneuron development is regulated by transcription factor modules that promote specific motoneuron characteristics and suppress specific interneuron characteristics. To deduce the logic by which transcriptional modules regulate motoneuron development, we propose to knock down expression of specific transcription factors using morpholino oligonucleotides and to examine motoneuron and interneuron markers, including axons, neurotransmitters, receptors, and ion channels. About half of the motoneurons initially formed during vertebrate development die from lack of availability or access to trophic support. We propose that the Notch and Netrin signaling pathways interact during motoneuron development, resulting in limited access of specific zebrafish motoneurons to trophic support and death of these cells. We propose to test this hypothesis by altering Netrin and Notch function. Many genes involved in motoneuron development remain unknown. We propose two screens to discover these genes: 1) A forward genetic screen using embryos with fluorescent motoneurons; this screen identifies genes based on function and 2) A microarray screen comparing spinal cords of embryos with supernumerary motoneurons to those of embryos lacking motoneurons; this screen indentifies genes based on differential expression. Genes these screens identify will feed back into the other studies in this proposal. Our ability to study individual zebrafish spinal neurons provides an unprecedented opportunity to learn the mechanisms that regulate motoneuron differentiation and survival. Our proposed studies will provide insights that should facilitate new therapies for treatment of motoneuron diseases and injury.